Summary Among all different types of DNA damages, double strand breaks (DSBs) are viewed as the most toxic ones that lead to genome instability. They are created by either endogenous agents such as reactive oxygen species, or exogenous ionizing radiation and chemicals. Unrepaired DSBs drive apoptosis and senescence, and incorrect DSB repair can lead to undesired genome rearrangements, such as deletions, translocations, and fusions. Non-homologous end-joining (NHEJ) pathway, in which the two broken DNA ends are directly ligated without referring to a homologous template, is the primary DSB repair pathway that remains active throughout the cell cycle. NHEJ is also responsible for the assembly of gene segments in V(D)J recombination, where various immunoglobulin genes are generated by exon recombination in immune cells. NHEJ is initialized by Ku heterodimer (Ku70/80) recognizing DSB ends. Upon recognizing a dsDNA broken end, Ku70/80 recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and assembles into the so-called DNA-PK holoenzyme. Other evolutionarily conserved NHEJ factors, including components of the ligase complex (DNA ligase IV, XRCC4 and XLF) are then recruited to the end reparation site. Successful DSB repair through NHEJ relies on the efficient bridging of two broken DNA ends, and this proposal aims to investigate the mechanism of NHEJ by directly visualizing the key steps of repair using single-particle cryo-EM. A more refined picture of the system specifically recognizing and correcting DSBs will provide an unprecedented, comprehensive view of these essential molecular machines during operation, and could lead to the development of novel treatments for various types of human cancer.